Project Summary A large body of functional amyloids has recently been discovered. However it remains unclear how these proteins can be regulated so that amyloid formation only occurs at the correct time and place. This study aims to answer this question by using the Orb2 functional amyloid as a model. Biological studies, as well as preliminary data from our lab, indicate that Orb2 forms amyloids in a two-step process that utilizes two separate cores: a seeding core that is used to trigger amyloid formation and secondary core that forms the completed amyloid. This two-step hypothesis presents a clear way to regulate amyloid formation by regulating the seeding core. This study will test this hypothesis through a series of solid-state nuclear magnetic resonance (ssNMR), electron paramagnetic resonance (EPR) and kinetics experiments on Orb2. ssNMR will be used to site specifically assign the main Orb2 fibril core and study its structure. EPR will provide additional structural information on this core and a potential seeding core in the Orb2 N-terminus. Kinetic, including EPR based, studies will show how the potential N-terminal core impacts amyloid formation. The model tested here, if correct, presents a viable mechanism for the regulation of numerous functional amyloids. This model is also relevant to human health and disease since understanding how controlled, functional amyloid formation occurs may help us understand how to prevent the formation of amyloids in disease.